Congenital heart defects are the most common type of birth defect, occurring in just less than 1% of all livebirths. One category of heart defects, heterotaxy, is thought to result from abnormal left-right patterning during embryogenesis. In its X-linked form, heterotaxy results from mutations in Zic3, a member of the Gli superfamily of transcription factors. Gli proteins are mediators of hedgehog signaling during development, and Zic3 is a candidate for a cell type specific trans-activator of hedgehog signaling at the node. The long term goal of this study is to define genetic and molecular interactions during L-R patterning that are required for cardiac morphogenesis. We have shown that mutations in ZIC3 result in abnormal subcellular localization in vitro, and that mice deficient in Zic3 recapitulate the phenotype identified in patients. Further, we have demonstrated that Zic3 acts upstream of a conserved signal transduction pathway acting at the embryonic node to control left-right patterning. In addition to Zic3's known expression in perinodal cells, we have recent data identifying expression in the cilia of the node. We hypothesize that the subcellular localization of Zic3 in the node and perinodal crown cells is critical for transduction of hedgehog signaling, determination of left- right patterning, and subsequent cardiac looping morphogenesis. The proposed experiments are designed to determine the tissue-, cell-type, and subcellular specific roles of Zic3 and examine the consequences of alteration of Zic3 expression on hedgehog signal transduction. Aim1 will test the cell autonomous requirement for Zic3 in perinodal crown cells for proper cardiac development and looping morphogenesis via a conditional loss of function approach. Aim 2 will test the hypothesis that alteration in the subcellular localization of Zic3 changes the net hedgehog signaling output from the node and lowers the threshold for cardiac and midline developmental defects. An understanding of the molecular hierarchy controlling cardiac looping is a necessary prerequisite for the development of genetic diagnostics and therapeutic interventions. These studies have the potential to identify molecular and genetic pathways contributing to cardiac development and will develop novel tools to dissect mechanisms underlying congenital heart disease.